The American Heart Association estimates that approximately 600,000 patients undergo heart bypass surgery and 400,000 have stents implanted to open blocked arteries. Approximately 6 million patients visit the emergency rooms each year because of suspected heart attacks. In each of these situations, the patients may be treated with angiogenesis-stimulating drugs. Thus, critical analysis in assessing the efficacy of these drugs and/or the disease state would provide a valuable tool to the clinician.
Endothelial cells are involved in the formation of blood vessels, or angiogenesis. This process is important for the growth of tumors and as a transport mechanism for circulating tumor cells (CTCs). Anti-angiogenesis drugs are in clinical development, either alone or in combination with traditional chemotherapeutic agents. Analysis of endothelial cells, after enrichment, using CELLTRACKS, a diagnostic imaging system, has applications in clinical trails as a tool to monitor efficacy as well as a tool for monitoring cancer patients receiving these drugs once they are marketed. Further, this type of analysis has applications in monitoring melanoma patients.
Circulating endothelial cells have been implicated in cardiovascular (such as assessing cardiac risk), inflammatory, and infectious diseases. Mature vascular endothelial cells exist in a variety of physiologic states ranging from quiescent to proliferative and activated to dysfunctional to terminal, whereupon they detach from the basement membrane and surrounding endothelial cells and enter the circulation. As the cause, fate and role of circulating endothelial cells (CECs) becomes better understood, the in vitro enumeration and characterization of CECs may offer a unique opportunity to study the vasculature and improve our understanding of a variety of homeostatic and disease processes. For instance, elevation of CECs has been observed in all of the above mentioned pathological conditions i.e. cancer, cardiovascular, inflammatory, infectious, and autoimmune disease (Mutin, M., Canavey, I., Blann, A., Bory, M., Sampol, J., Dignat-George, F. Direct evidence of endothelial injury in acute myocardial infarction and unstable angina by demonstration of circulating endothelial cells. Blood, 93: 2951-2958, 1999; also see WO 2004/045517). In particular, cancer CEC levels may increase due to active tumor angiogenesis, vascular damage as a result of tumor apoptosis/necrosis or as a side effect of therapy on non-tumor vasculature. However, the lack of standardized assay methods, the lack of consensus on the definition of a CEC (Beerepoot, L. V., Mehra, N., Vermaat, J. S. P., Zonnenberg, B. A., Gebbink, M. F. G. B., Voest, E. E. Increased levels of viable circulating endothelial cells are an indicator of progressive disease in cancer patients. Annals of Oncology, 15: 139-145, 2004) and disease heterogeneity have led to a wide variation in the reported ranges of CECs (1 to 5700 per mL). All of these factors make interpretation and comparison of existing studies quite difficult if not impossible.
Surface antigens of vascular endothelial cells has come from several lines of research, including studies of lymphocyte homing, inflammation, blood clotting, and tumor metastasis. Monoclonal antibodies (mAbs) have proven to be valuable tools for dissecting the antigenic structure of endothelial cells in different organs, tissues or segments of the vascular system, and the endothelial responses to inflammation, tissue damage, and tumor growth. Furthermore, mabs have been used in the biochemical and molecular genetic characterization of endothelial antigens and in the functional analyses of endothelial molecules in vitro and in vivo.
Several categories of endothelial antigens have been distinguished, based on their distribution patterns in normal and lesion blood vessels. These include (i) antigens with wide distribution in the vascular system, such as Factor VIII-related antigen; (ii) antigens restricted to vessels in specific organs or tissues, or to unique histologic types of vessels, as illustrated by vascular addressins and GlyCAM-1; and (iii) inducible antigens, such as E-selectin, VCAM-1, and ICAM-1, that are not present or expressed at low levels in normal endothelium but are upregulated in inflamed tissues in vivo and/or induced or cultured endothelial cells by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1).
Many laboratory and clinical procedures employ bio-specific affinity reactions for isolating rare cells from biological samples. Such reactions are commonly employed in diagnostic testing, or for the separation of a wide range of target substances, especially biological entities such as cells, proteins, bacteria, viruses, nucleic acid sequences, and the like.
Various methods are available for analyzing or separating target substances based upon complex formation between the substance of interest and another substance to which the target substance specifically binds. Separation of complexes from unbound material may be accomplished gravitationally, e.g. by settling, or, alternatively, by centrifugation of finely divided particles or beads coupled to the target substance. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immune and other bio-specific affinity reactions. See, for example, U.S. Pat. No. 4,554,088. Generally, any material that facilitates magnetic or gravitational separation may be employed for this purpose. However, it has become clear that magnetic separation means are the method of choice.
Small magnetic particles of the type described above are quite useful in analyses involving bio-specific affinity reactions, as they are conveniently coated with biofunctional polymers (e.g., proteins), provide very high surface areas and give reasonable reaction kinetics. Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunological reagents.
High gradient magnetic separation with an external field device employing highly magnetic, low non-specific binding, colloidal magnetic particles is the method of choice for separating a cell subset of interest from a mixed population of eukaryotic cells, particularly if the subset of interest comprises but a small fraction of the entire population. Such materials, because of their diffusive properties, readily find and magnetically label rare events, such as tumor cells in blood. For magnetic separations for tumor cell analysis to be successful, the magnetic particles must be specific for epitopes that are not present on hematopoeitic cells.
In summary, a useful diagnostic test needs to be very sensitive and reliably quantitative. A system that provides a consistent, reliable, and reproducable enumeration of target endothelial cells is needed in diagnostic and prognostic analysis of cardiovascular monitoring. A blood test that detects a single or a few endothelial or fungal cells in less than 30 ml of blood would provide a very sensitive and early detection mechanism in disease assessment.